Re: Prot. Pur: Interleukins
gsarath@unlnotes.unl.edu
Fri, 27 Aug 1999 18:08:47 -0500
Hi Scottie: I just had to write. Being a protein-type, I have generally avoided
tags, partly because of problems during crystallization and partly because of
biochemical considerations (also because of my enduring love for frustrating
purification schemes). I have never worked with interleukins, but, if they are
smaller proteins (15 to 18 kDa), a first step could utilize phenyl-sepharose
(equilibrated with 1 to 1.8 M ammonium sulfate in buffer of choice), these
smaller molecules should wash off with 0.9 to 0.4 M ammonium sulfate) leaving
most of the FBS and associated junk stuck on the column. I guess, the simplest
assay would be to use SDS-PAGE of desalted extracts (centricon-10). The
enriched fraction can be concentrated and applied to a Superose-12 column in
buffer + 0.1 M NaCl). You should acheive some enrichment this way (if the gods
smile - even good enrichment). For most crude purifcation, the fastflow
Pharmacia hi-load phenyl-sepharose (packed even in a small glass column) works
very well. If there is a need for further purification (you might get efficient
enrichment at the first step by using a fractional ammonium sulfate cut - say 0
to 60% to get rid of a lot of FBS proteins and a 60 to 90% to enrich for IL,
then solubilize this pellet in 1.8M ammonium sulfate and apply to the
Phenyl-sepharose column). If you know the solution pI of these beasties, you
could take the Phenyl-sepharose fraction, buffer-exchange and chromatograph on
an ion-exchange column.
In any case, one bulk step with a finer-resolution step should yield adequate
purification for many general uses. Good luck, Gautam